Abstract
The fluorescent reporter enhanced Green Fluorescent Protein (EGFP) has been used for assaying a wide range of biological activities ranging from gene expression, or localization of target proteins through to intermolecular interactions. However, over-production of this protein in Escherichia coli has resulted in the presence of inclusion bodies, which complicates recovery of the protein in significant quantities. In this paper, we describe a single-step method for isolating the protein from a Glutathione-S-Transferase (GST) fusion protein, release of the EGFP protein from the fusion was demonstrated using a biotinylated variant of Human Rhinovirus 14 3C protease that we have also constructed. We also suggest the potential uses of the biotinylated protease for bionanotechnology and synthetic biology.
Copyright © 2011 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3C Viral Proteases
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Biotechnology / methods*
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Biotinylation
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / isolation & purification
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Cysteine Endopeptidases / metabolism*
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Escherichia coli
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Glutathione Transferase / genetics
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Glutathione Transferase / metabolism*
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / isolation & purification*
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Green Fluorescent Proteins / metabolism
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Humans
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Immunoblotting
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Plasmids / chemistry*
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Plasmids / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification*
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Recombinant Fusion Proteins / metabolism
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Rhinovirus / enzymology
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Solubility
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Spectrometry, Fluorescence
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Synthetic Biology / methods*
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Viral Proteins / genetics
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Viral Proteins / isolation & purification
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Viral Proteins / metabolism*
Substances
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Recombinant Fusion Proteins
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Viral Proteins
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enhanced green fluorescent protein
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Green Fluorescent Proteins
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Glutathione Transferase
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Cysteine Endopeptidases
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3C Viral Proteases